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This summary article presents an overview of the molecular relationships among the voltage-gated sodium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the sodium channel family can be found at .
Two mechanisms of Na+ influx have been observed using synaptosomal plasma membrane vesicles purified by density gradient centrifugation from a synaptosomal hypotonic lysate. First, a 5-fold increase in uptake over basal Na+ entry occurs with 50 microM veratridine. The veratridine-dependent Na+ uptake is partially inhibited by 2 microM tetrodotoxin with an apparent time dependency of action (half-maximal inhibition in approximately 20 min). Second, a larger Na+ accumulation (approximately 15-fold above basal) was observed with 2.5 mM ATP, this effect being dependent on internal K+ loading of vesicles although inhibited by high external K+. The two uptake processes are believed to represent operation of the plasma membrane voltage-sensitive Na+ channel, and the Na+-pumping (Na+ + K+)-ATPase, respectively. Both Na+ flux mechanisms appear to operate in a single population of vesicles since opening of the Na+ channel with veratridine diminishes the ATP-dependent accumulation of Na+ by over 75%. An inverted orientation of the plasma membrane vesicles is likely to account for the functioning of the ATP-dependent Na+ pump and may also account for the low sensitivity and time dependency of the inhibitory action of tetrodotoxin on Na+ channel-opening. Na+ accumulated by the Na+ pump was rapidly effluxed by 10 mM external Ca2+ via the Na+-Ca2+ exchange mechanism which (together with an ATP-dependent Ca2+-accumulating mechanism) was recently characterized in the vesicles (Gill, D. L., Grollman, E. F., and Kohn L. D. (1981) J. Biol. Chem. 256, 184-192). This result, together with the observed inhibition of Ca2+ influx via (Na+-Ca2+)-exchange due to veratridine-mediated Na+ flux, strongly suggests that the Na+ pump, Na+ channel, and both Ca2+ transport mechanisms function in a single population of inverted plasma membrane vesicles.
Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn2+ and Cd2+. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn2+ and Cd2+, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn2+ or Cd2+, but this mutation markedly reduces sensitivity to block by Ca2+ and Co2+. The double mutant channel (F385C.N388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.
Sodium enters tight epithelia across the apical plasma membrane through a sodium channel, a process inhibited by submicromolar concentrations of amiloride and benzamil. Using membrane vesicles from bovine kidney cortex, we found that sodium transport through the sodium channel was inhibited by benzamil with an IC50 of 4 nM. Amiloride (IC50 = 400 nM) was a weaker inhibitor of sodium transport. [3H]Benzamil bound to the vesicles at a single class of high affinity binding sites with a Kd of 5 nM, the similarity of which to the IC50 suggests that these binding sites are associated with the sodium channel. Amiloride displaced bound [3H]benzamil with a Ki of 2,500 nM. Bromobenzamil is a photoactive amiloride analog with potency similar to benzamil in inhibiting sodium transport (IC50 = 5 nM) and binding to the sodium channel (Kd = 6 nM). [3H]Bromobenzamil was specifically photoincorporated into three molecular weight classes of polypeptides with apparent Mr values of 176,000, 77,000, and 47,000. The photoincorporation of [3H]bromobenzamil into these three classes of polypeptides was blocked by addition of excess benzamil and by amiloride in a dose-dependent manner. These data suggest that these polypeptides are components of the epithelial sodium channel.
1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.
Sodium channels from rat brain, rat skeletal muscle, chick brain, and eel electroplax were compared by using the mu-conotoxins GIIIA, PIIIA, and StIII and [3H]saxitoxin. Rat skeletal muscle and eel electroplax sodium channels are equally sensitive to GIIIA, PIIIA, and StIII, displacing >90% of the [3H]saxitoxin binding sites in rat skeletal muscle and eel electroplax membranes and exhibiting inhibitory concentrations at half-maximal percentage specific binding (IC50) of 0.97 nM for GIIIA, 1.3 nM for PIIIA in rat skeletal muscle, and concentrations of 3.5 nM for GIIIA and 2.8 nM for PIIIA in eel electroplax. PIIIA and GIIIA at all concentrations inhibit only up to 10% of [3H]saxitoxin binding sites in chick brain membranes. Mu-conotoxin StIII at all concentrations inhibits only up to 10% of [3H]saxitoxin binding sites in rat brain membranes and displays two-site binding inhibition of the [3H]saxitoxin binding sites in rat skeletal muscle. PIIIA displaces >60% of the [3H]saxitoxin binding sites (IC50 of 44 nM) while GIIIA blocks out only 30% of the binding sites in rat brain sodium channels (IC50 of 69 nM). Thus, sodium channel subtypes can be classified into two categories: mu-conotoxin sensitive (i.e., subtypes predominantly found in rat skeletal muscle and eel electroplax) and insensitive (i.e., subtypes predominantly found in rat and chick brain).
Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind alpha- and alpha-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with alpha-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled alpha-scorpion toxins AaH II and LqhalphaIT, the most active alpha-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The alpha-mammal scorpion toxins and the anti-insect Lqh alphaIT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with alpha-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and Lqh alphaIT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster.
Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to the protease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodium channel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.
Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.
Sodium channels from human brain tissue were incorporated into voltage-clamped planar lipid bilayers in presence of batrachotoxin and exposed to increasing concentrations of the intravenous anaesthetic drug etomidate (0.03-1.02 mM). Etomidate interacted with the sodium-conducting pathway of the channel causing a concentration-dependent block of the time-averaged sodium conductance (computer fit of the concentration-response curve: half-maximal blocking concentration, EC50, 0.19 mM; maximal block, block(max), 38%). This block of sodium-conductance resulted from two distinct effects (I) major effect: reduction of the sodium-channel amplitude and (II) minor effect: reduction of the fractional channel open-time. These results were observed at concentrations above clinically-relevant serum concentrations (up to 0.01 mM), suggesting only a limited role for human brain sodium channels in the mechanism of action of etomidate during clinical anaesthesia.
Sodium channel drugs were used to modulate the anesthetic effects of bupivacaine in mice. Anesthesia was measured following perisciatic injection of bupivacaine with vehicle or neurotoxin in the popliteal region. The site 1 Na(+) channel blocker tetrodotoxin alone was inactive, but increased the anesthetic effects of bupivacaine. We hypothesized that the site 2 and site 3 Na(+) channel openers veratridine and anemone toxin II (ATXII), respectively, would antagonize bupivacaine. Paradoxically, both drugs enhanced bupivacaine. In bupivacaine-treated mice, a significant correlation was observed between limb weakness scores and paw withdrawal latencies. The correlation coefficients were higher when tetrodotoxin, veratridine, or ATXII was coadministered with bupivacaine. In conclusion, veratridine and ATXII may have increased the stimulus-dependent binding of bupivacaine to Na(+) channels, thereby increasing the anesthetic effects of bupivacaine.
Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-S6). The highly charged S4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in S4 segments of each domain of the human heart Na+ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na+ current, consistent with a role of S4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (eo) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h infinity) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in S4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.
Tertiary amine local anesthetics (LAs) are clinically valuable agents for controlling pain and for treating some cardiac arrhythmias. These drugs inhibit conduction of electrical activity by blocking voltage-gated sodium channels. Interestingly, LAs can influence the conduction of electrical activity in heart muscle without markedly altering normal skeletal muscle activity. This review discusses the interactions between sodium channels and LAs, the methods used to investigate these interactions, and the mechanisms proposed to explain the greater LA sensitivity of cardiac sodium channels as compared with skeletal muscle sodium channels.
Sodium channels encoded by the rat IIA cDNA clone [Auld, V. J., Goldin, A. L., Krafte, D. S., Marshall, J., Dunn, J., Catterall, W. A., Lester, H. A., Davidson, N. & Dunn, R. J. (1988) Neuron 1, 449-461] differ at seven amino acid residues from those encoded by the rat II cDNA [Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. & Numa, S. (1986) Nature (London) 320, 188-192]. When expressed in Xenopus oocytes, rat IIA channels display a current-voltage relationship that is shifted 20-25 mV in the depolarizing direction relative to channels expressed from rat II cDNA or rat brain poly(A)+ mRNA. By modifying each variant residue in rat IIA to the corresponding residue in rat II, we demonstrate that a single Phe----Leu substitution at position 860 in the S4 segment of domain II is sufficient to shift the current-voltage relationship to that observed for channels expressed from rat brain poly(A)+ RNA or rat II cDNA. Rat genomic DNA encodes leucine but not phenylalanine at position 860, indicating that the phenylalanine at this position in rat IIA cDNA likely results from reverse transcriptase error.
Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.
TWO SODIUM TRANSPORT SYSTEMS HAVE BEEN ANALYZED IN THIS WORK: the voltage-sensitive sodium channel and the (Na(+), K(+)) ATPase pump. The sodium channel has been studied using a tritiated derivative of tetrodotoxin; the sodium pump has been studied using tritiated ouabain. Properties of interaction of tritiated tetrodotoxin and of tritiated ouabain with their respective receptors were observed in normal human skeletal muscle and in muscles of patients with myotonic muscular dystrophy and with lower motor neuron impairment. Levels of sodium pump and of sodium channels were measured at different stages of membrane purification. Microsomal fractions of normal human muscle have maximal binding capacities for tetrodotoxin of 230 fmol/mg of protein and of 7.4 pmol/mg of protein for ouabain. Dissociation constant for the complexes formed by the tetrodotoxin derivative and by ouabain with their respective receptors were 0.52 nM and 0.55 muM, respectively. In muscles from patients with myotonic muscular dystrophy, the maximal binding capacity for tetrodotoxin, i.e., the number of Na(+) channels was found to be very similar to that found for normal muscle. The maximal binding capacity for ouabain, i.e., the number of Na(+) pumps was three- to sixfold lower than in normal muscle. Dissociation constants for the complexes formed with the tetrodotoxin derivative and with ouabain were the same as for normal muscle. In muscles from patients with lower motor nerve impairment, the maximal binding capacities for tetrodotoxin and for ouabain were twice as high as in normal muscle. Again, dissociation constants for the complexes formed with the tetrodotoxin derivative and with ouabain were nearly unchanged as compared with normal muscle. These results suggest that sodium transport systems involved in the generation of action potentials and/or in the regulation of the resting potential are altered both in myotonic muscular dystrophy and in lower motor neuron impairment.
Whole-cell gating current recording from rat brain IIA sodium channels in Xenopus oocytes was achieved using a high-expression system and a newly developed high-speed two-electrode voltage-clamp. The resulting ionic currents were increased by an order of magnitude. Surprisingly, the measured corresponding gating currents were approximately 5-10 times larger than expected from ionic permeability. This prompted us to minimize uncertainties about clamp asymmetries and to quantify the ratio of sodium permeability to gating charge, which initially would be expected to be constant for a homogeneous channel population. The systematic study, however, showed a 10- to 20-fold variation of this ratio in different experiments, and even in the same cell during an experiment. The ratio of P(Na)/Q was found to correlate with substantial changes observed for the sodium reversal potential. The data suggest that a cytoplasmic sodium load in Xenopus oocytes or the energy consumption required to regulate the increase in cytoplasmic sodium represents a condition where most of the expressed sodium channels keep their pore closed due to yet unknown mechanisms. In contrast, the movements of the voltage sensors remain undisturbed, producing gating current with normal properties.
The voltage-dependent properties of the voltage-activated sodium channel were studied in neonatal (1-2-day-old) and adult rat ventricular cardiac myocytes using the whole-cell variation of the patch-clamp technique (16 degrees C, [Na]i = 15 mM, [Na]o = 25 mM). The voltage dependence of the sodium conductance-membrane potential relation was similar in both neonatal and adult myocytes except for a difference in slope; the adult sodium conductance-membrane potential relation was slightly more steep. Neonatal cells also differed from adult cells by demonstrating a more negative voltage midpoint of their sodium availability curve, a slower rate of recovery from inactivation at hyperpolarized potentials, and a greater extent of slow inactivation development compared with adult cells. Phenytoin (40 microM) reduced the sodium current in a tonic and use-dependent manner in both adult and neonatal myocytes. However, phenytoin (40 microM) produced significantly more tonic block at negative holding potentials (e.g., -140 mV) in neonatal myocytes (22 +/- 5% [mean +/- SEM], n = 14) than in adult myocytes (10 +/- 2%, n = 11) (p less than 0.05). The amplitudes of use-dependent block obtained during trains of 1-second pulses to -20 mV were also significantly greater in neonatal myocytes than in adult myocytes when the diastolic interval was varied over a range of 0.1-1.5 seconds (p less than 0.05). Definition of the time courses of block development at -20 mV indicated that phenytoin had a slightly higher affinity for inactivated sodium channels in neonatal cells. In addition, the time constant of recovery from use-dependent block by phenytoin was found to be significantly longer in neonatal cells than in adult cells at membrane potentials between -160 and -100 mV (p less than 0.001). The marked differences in phenytoin effect on cardiac sodium channels in neonatal versus adult rat cardiac myocytes suggest that there may be significant developmental changes in the sodium channel blocking effects of class I antiarrhythmic drugs in cardiac tissue.